Project Summary Our 4-yr project aims to develop a new ultrasonic Doppler method named Higher-Order Perfusion Estimation (HOPE) imaging. Applications enabled by this technology are routine assessments of microvascular disease progression in diabetic patients with symptoms of peripheral artery disease (PAD). Technical advances include new ultrasonic echo sampling and filtering techniques that significantly increase the sensitivity and specificity of standard sonographic instruments to spatially disorganized patterns of red-blood-cell movement without the addition of contrast media. Sensitivity to slowly perfusing blood is increased by transmitting a sparse regular sequence of Doppler pulses over long durations (1-10s). To counter a concomitant increase in clutter-signal power in perfusing-blood frequency channels, spatiotemporal echo acquisitions are first rearranged into 3-D data arrays and a 3-D singular-value decomposition (3D-SVD) clutter filter is formed for each experiment. Our approach now successfully separates weak perfusing-blood echoes from other echo-signal sources in the peripheral vasculature because of the typically narrow eigen-bandwidth of clutter echoes in peripheral muscle. Preliminary results using echo simulation, microchannel flow phantoms, and preclinical models of mouse ischemic hindlimb and melanoma lesions demonstrate that our method is very well designed for monitoring steady peripheral microvascular flow patterns using commercial ultrasonic instruments (with software updates). Four aims are proposed to demonstrate the utility of HOPE imaging (both power and color-flow) for measuring blood flow and perfusion. Aim 1 expands preliminary studies in mouse models to evaluate perfusion measurement sensitivity at 12-24 MHz in diabetic animals, and to uncover mechanisms of the angiogenic response of tissues to sudden ischemia. Aim 2 continues development of HOPE imaging by improving perfusing- blood echo sensitivity and clutter-filter performance under more general imaging conditions (viz., broad eigen- bandwidth clutter and 3-D spatially varying perfusion). Aim 3 focuses on a progressively ischemic pig model using 5-12 MHz HOPE imaging, MR angiography, and radioactive microsphere techniques to calibrate and compare HOPE imaging results with standard clinical approaches. Aim 4 is a 4-yr, 150-patient study designed to evaluate the diagnostic performance of HOPE imaging at assessing PAD. The overall project aims to develop existing ultrasonic instruments into highly-effect tools for evaluating microvascular changes leading to common disabilities and major cardiovascular events. The three in vivo studies proposed in this plan are designed to develop and evaluate HOPE imaging specifically for PAD diagnosis, staging, and therapeutic monitoring.